Bms ccr2 22

Manufactured by Bio-Techne

Sourced in United Kingdom

The BMS CCR2 22 is a laboratory equipment product manufactured by Bio-Techne. It is designed to assist in scientific research and analysis. The core function of this product is to perform a specific task or measurement, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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5 protocols using bms ccr2 22

Sialidase Treatment and Chemokine Receptor Blocking for CD8+ T Cell Analysis

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For removing sialic acid residues, cell-sorted subsets of CD8⁺ T cells were treated with 0.1 units of sialidase (from Vibrio cholera, Sigma-Aldrich) in 1 ml RPMI 1640 (Life Technologies) containing 10% FBS (Gemini Bio-Products) and 2 mM L-glutamine for 2.5 hr at 37°C. For inhibiting G_i/o proteins, CD8⁺ T cells were pre-incubated with pertussis toxin (1 μg/ml (R and D Systems) in RPMI 1640 medium containing 10% FBS and 10 mM HEPES for 3 hr at 37°C. For blocking CCR2 and CCR5, pre-incubation was with BMS CCR2 22 (2 μM (Tocris, Minneapolis, MN) or Maraviroc (10 μM (Tocris), respectively, for 30 min at 37°C and inhibitors were left in the medium throughout the assay. For neutralizing CCL20, HUVEC monolayers in flow chambers were pre-treated for 2 hr at 37°C with 20 μg/ml anti-human CCL20/MIP-3α antibody (c67310; R and D Systems), and antibody was maintained at 10 ng/ml throughout the assay.

Lee C.H., Zhang H.H., Singh S.P., Koo L., Kabat J., Tsang H., Singh T.P, & Farber J.M. (2018). C/EBPδ drives interactions between human MAIT cells and endothelial cells that are important for extravasation. eLife, 7, e32532.

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Neuroinflammation Modulators Protocol

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Recombinant rat CCL2 was obtained from R&D Systems (Minneapolis, MN, USA). Minocycline was purchased from Sigma (St Louis, MO, USA). BMS CCR2 22, picrotoxin, strychnine, tetrodotoxin, 2-amino-5-phosphonovalerate (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Tocris Bioscience (Bristol, UK). WP9QY was purchased from Santa Cruz (Dallas, TX, USA).

Huang C.Y., Chen Y.L., Li A.H., Lu J.C, & Wang H.L. (2014). Minocycline, a microglial inhibitor, blocks spinal CCL2-induced heat hyperalgesia and augmentation of glutamatergic transmission in substantia gelatinosa neurons. Journal of Neuroinflammation, 11, 7.

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Pharmacological Modulation of P2Y Receptors

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All chemicals were purchased from Sigma-Aldrich (UK) with the exception of BMSCCR222, BAPTA AM, MRS2578, ARL67156, AR-C66096 and Bordetella pertussis toxin (PTx; Tocris Bioscience, UK), MRS2179, MRS2211 (Abcam Biochemicals, UK), CCL2, TNFα and Fluo-4 AM (Life Technologies, UK), and Calcein AM (Santa Cruz Biotechnology, Germany). THP-1 and HEK 293T cells were procured from the European Collection of Cell Cultures (ECACC), and HUVECs from Caltag Medsystems (UK). Human 1321N1 P2Y₆ stable cells were a kind gift from Jens Leipziger (Aarhus University, Denmark). Compounds used did not induce toxicity under assay conditions as determined by a Trypan Blue exclusion assay or lactate dehydrogenase (LDH) release assay.

Campwala H., Sexton D.W., Crossman D.C, & Fountain S.J. (2014). P2Y6 receptor inhibition perturbs CCL2-evoked signalling in human monocytic and peripheral blood mononuclear cells. Journal of Cell Science, 127(22), 4964-4973.

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Monocyte Tracking in Mouse Models

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mPEG-DSPE and NHS-PEG-DSPE were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Mouse CCR2 antibody (Clone # 475301) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA) and had a molecular weight of 593.66 Da. Both 1,1-dioctadecyl-3,3,3,3,-tetram-ethylindodicarbocyanide (DiD) and DAPI were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The CCR2 antagonist BMS CCR2 22 was purchased from Tocris Bioscience (Bristol, UK). Anti-NK1.1-biotin, anti-CD49b-biotin, anti-Ly6G-biotin, anti-MHC II-biotin, anti-CD90.2-biotin, anti-CD45R (B220)-biotin, anti-F4/80-APC, anti-CD11c-PE, and anti-Ly6C-fluorescein isothiocyanate were purchased from Miltenyi Biotec (Auburn, CA, USA). Anti-CD11b-BV421 and V500 streptavidin beads were purchased from BD Biosciences (San Jose, CA, USA), col-lagenase II was purchased from Worthington (Lakewood, NJ, USA), and DNase I was from Thermo Fisher Scientific (Waltham, MA, USA). The RAW 264.7 cells were purchased from American Type Culture Collection. All other chemicals and solvents were purchased from Thermo Fisher Scientific unless otherwise stated.

Wang J., Seo M.J., Deci M.B., Weil B.R., Canty J.M, & Nguyen J. (2018). Effect of CCR2 inhibitor-loaded lipid micelles on inflammatory cell migration and cardiac function after myocardial infarction. International Journal of Nanomedicine, 13, 6441-6451.

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Depletion of Monocyte Chemotactic Chemokines

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To deplete single or a combination of different monocyte chemotactic chemokines, biotinylated anti-human antibody against CCL2, CCL3, CCL4, and CCL8 were added at a concentration of 5 μg/mL to the different CoM. The biotinylated antibodies were incubated with the CoM for one hour at room temperature before addition of magnetic streptavidin beads (Dynabeads MyOne Streptavidin T1, Life Technologies) using 10 9 beads/mL. These were incubated for one hour with the CoM before removing the beads magnetically.
Successful depletion was confirmed by ELISA using paired capture and detection antiboides for each chemokine (eBioscience) according to manufacturer's instructions and analysed using a Multiskan absorbance plate reader (Thermo Labsystems). For chemokine receptor targeting, 5x10 6 cells/mL PBMC were incubated with inhibitors of CCR2 (RS 504393 or BMS CCR2 22, both from Tocris), CCR5 (Maraviroc, Tocris), or CXCR4 (AMD 3465 hexahydrobromide, Tocris), used at 10 nM, for 30 minutes.

Lapp T., Zaher S.S., Haas C.T., Becker D.L., Thrasivoulou C., Chain B.M., Larkin D.F, & Noursadeghi M. (2015). Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea. Investigative ophthalmology & visual science, 56(12).

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